Isotachophoretic focusing of nucleic acids

ABSTRACT

A method and system are presented for fast and efficient isolation, purification and quantitation of nucleic acids from complex biological samples using isotachophoresis in microchannels. In an embodiment, a sieving medium may be used to enhance selectivity. In another embodiment, PCR-friendly chemistries are used to purify nucleic acids from complex biological samples and yield nucleic acids ready for further analysis including for PCR. In another embodiment, small RNAs from biological samples are extracted, isolated, preconcentrated and quantitated using on-chip ITP with a high efficiency sieving medium. The invention enables fast concentration and separation (takes 10s to 100s of seconds) of nucleic acids with high selectivity and using lower volumes of reagents (order of 10s of μL to focus less than 1 pg/μL of nucleic acid).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication No. 61/209,199, filed Mar. 3, 2009, which is herebyincorporated by reference in its entirety.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with Government support under ContractN01-HV-28183awarded by National Institues of Health. The Government hascertain rights in this invention.

REFERENCE TO SEQUENCE LISTING, COMPUTER PROGRAM, OR COMPACT DISK

Applicants assert that the text copy of the Sequence Listing isidentical to the Sequence Listing in computer readable form found on theaccompanying computer file. Applicants incorporate the contents of thesequence listing by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of isotachophoresis (ITP),particularly for selective separation, detection, extraction,pre-concentration or quantitation of RNA, DNA, and/or other biologicalmolecules.

2. Related Art

Presented below is background information on certain aspects of thepresent invention as they may relate to technical features referred toin the detailed description, but not necessarily described in detail.That is, individual parts or methods used in the present invention maybe described in greater detail in the materials discussed below, whichmaterials may provide further guidance to those skilled in the art formaking or using certain aspects of the present invention as claimed. Thediscussion below should not be construed as an admission as to therelevance of the information to any claims herein or the prior arteffect of the material described.

Microfluidics has become an alternative to traditional techniques forbiological and medical analysis and offers the use of small reagentvolumes, fast analyses, and the potential for parallelization.1Polymerase chain reaction (PCR), capillary electrophoresis, 3immunoassays,4 and many other analytical techniques used in biology andmedicine have been successfully miniaturized. However, samplepreparation is often still a challenge and a limiting factor in thecapability of many devices, so that most miniaturized systems have usedprepurified, ideal samples as analyte. One important application is thepurification of nucleic acids (NA) from complex biological samples,i.e., a complex mixture of macromolecules, which may contain smallmolecules as well. We here demonstrate a simple, fast, efficient, andsensitive technique for the purification of NA from whole blood whichleverages the physicochemistry of isotachophoresis (ITP). The standardmethod for NA purification is based on solid phase extraction (SPE). Forexample, commonly used QIAGEN (Valencia, Calif.) purification columnsrely on the adsorption of NA on silica membranes.6 Extensive work byLanders and co-workers has shown successful microchip integration of SPEwith application to purification of DNA7 and RNA8 and successfulintegration with on-chip PCR. While micro-SPE shows excellent efficiencyand throughput, the process requires specialized materials andfabrication (e.g., micropillars or packing of silica beads). Further,the typical SPE protocol involves three successive steps (loading,washing, elution), requires bulk flow control, and uses a PCR inhibitingchemistry (e.g., chaotropic agents, organic solvents). Another exampleof SPE is the Quick Gene Mini-80, a nucleic acid extraction device byFujifilm Life Sciences which uses pressurized filtration accompanied bywashing and eluting steps to isolate nucleic acids.

ITP is a well-established separation and preconcentration technique. Itleverages a heterogeneous buffer system to generate strong electricfield gradients, allowing simultaneous focusing and separation of ionicspecies based on their effective electrophoretic mobilities. ITP hasbeen marginally used as a sample purification method. For instance,Caslayska et al. (Caslayska, J.; Thormann, W. J., Chromatogr., A 1992,594, 361-369) used ITP to simultaneously purify and isolate proteins.Kondratova et al (Kondratova, V. N.; Serd'uk, O. I.; Shelepov, V. P.;Lichtenstein, A., Biotechniques, 2005, 39, 695-699) concentrated andisolated extracellular DNA from blood plasma and urine by agarose gelITP with applications to cancer diagnosis. This ITP isolation procedureyields DNA in an agarose gel slab which requires further purificationsteps prior to analysis. To our knowledge, ITP has never been applied tosample preparation from biological samples for analysis. The method inour invention is capable of accepting into the on-chip process a complexbiological sample like whole blood added directly to the ITP wellwithout pre-processing like centrifugation or filtration.

Presented below is an ITP-based purification method for extractinggenomic DNA from a biological sample such as whole blood lysate as asample. Previous work has been directed towards methods in which acomplex sample such as this, containing a mixture of macromolecules, ispre-treated prior to ITP. In addition to separating Genomic DNA fromcellular components such as membranes, organelles, proteins, and othernucleic acids.

In addition, the present methods relate to separation of small RNAs fromother nucleic acids (sometimes referred to as polynucleic acids, asopposed to single nucleotides).

Small RNAs are involved in RNA interference (RNAi).

RNAi refers to the regulation of gene expression, in which small RNAsmediate gene silencing. In the RNAi process small RNAs are loaded ontoArgonaute proteins at the core of an RNA-induced silencing complex(RISC), where these noncoding RNAs guide the sequence-specific silencingof transcripts through base-pairing interactions. The transcripts aretypically messenger RNAs (mRNAs), which are cleaved or prevented frombeing translated by ribosomes, leading to their degradation. In humansat least 30% of the genes are thought to be regulated by miRNAs, whichtune protein synthesis from thousands of genes. Further, miRNAs haverecently been linked with common diseases.

miRNA expression profiling has been done using Northern blotting butthis technique involves laborious, time-consuming procedures and lacksautomation. The method of reverse transcription polymerase chainreaction (RT-PCR) is typically restricted to the quantitation ofspecifically lengthened miRNAs or pre-miRNAs, because the short lengthof miRNAs significantly limits the flexibility of primer design.Microarrays allow profiling miRNAs in a highly efficient parallelfashion, but this technique has encountered difficulties in reliablyamplifying miRNAs without bias.

To overcome the obstacle of selective and sensitive quantitation devicesfor miRNA research, various techniques have been developed: a nanogappedmicroelectrode-based biosensor array; electrocatalytic nanoparticle tagsand gold nanoparticle probes; and capillary electrophoresis with thesieving matrices of poly(ethylene oxide) or poly(vinyl pyrrolidone) formiRNAs, and poly(ethylene glycol) for general oligonucleotidesapplications. Compared to conventional capillary electrophoresis,microchip electrophoresis techniques offer considerably shorter analysistimes, the ability to work with small sample volumes, and theopportunity of combination with additional on-chip assay steps. However,the loading of microchannels with gels remains challenging due to thehigh viscosity of crosslinked gels and consequential bubble formation.

Thus there exists a need in the field for fast and efficient method forextraction, isolation, preconcentration and quantitation of nucleicacids including small RNAs from complex biological samples like blood,blood lysate, cell culture, cell lysates, etc. For the presentinvention, we have extracted and purified nucleic acids from complexbiological samples using ITP in microchannels and with PCR-friendlychemistries. We have extracted, isolated, preconcentrated andquantitated small RNAs from complex biological samples using ITP inmicrochannels loaded with a high efficiency sieving medium.

SPECIFIC PATENTS AND PUBLICATIONS

Jung et al., “On-Chip Millionfold Sample Stacking Using TransientIsotachophoresis,” Anal. Chem., 78:2319-2327 (2006) discloses on-chipITP integrated with on chip capillary electrophoresis.

Persat et al., “Purification of Nucleic Acids from Whole Blood UsingIsotachophoresis,” Anal. Chem., 81:9507-9511 (2009), published on theweb Oct. 15, 2009, discloses certain work described here.

Khurana and Santiago, “Preconcentration, Separation, and IndirectDetection of Nonfluorescent Analytes Using Fluorescent MobilityMarkers,” Anal. Chem., 80:279-286 (Nov. 22, 2007) discloses a techniquewhich uses ITP for both preconcentration and separation. The authorsemploy a leading electrolyte (LE), trailing electrolyte (TE), and a setof fluorescent markers of mobilities designed to bound those ofnonfluorescent analytes of interest.

US 2002/0079223 by Williams et al., entitled “TandemIsotachophoresis/zone Electrophoresis Method and System,” discloses amicrofluidic device which may be used in ITP.

WO 2008/053047 by Weber et al., entitled “Novel methods, kits anddevices for isotachophoresis applications,” published 8 May 2008,discloses a method and device where the use of at least one T medium, atleast one diluted T medium and at least one L medium according to thepresent invention provides an essentially constant pH over the wholewidth of the separation zone between the electrodes of an apparatussuitable to carry out an ITP separation.

Prest et al., “Miniaturised isotachophoresis of DNA,” J. Chromatog.,1156:154-159 (2007) discloses an electrolyte system comprising a leadingelectrolyte of 5 mMperchloric acid at pH 6.0 and a terminatingelectrolyte of 10 mM gallic acid used to perform isotachophoresis of DNAcontaining samples on a miniaturised poly(methyl methacrylate) device.Under such conditions it was found that no separation of DNA fragmentswas observed with the substance migrating instead as a singleisotachophoretic zone.

Kondratova et al., “Concentration and isolation of DNA from biologicalfluids by agarose gel isotachophoresis,” BioTechniques, 39:695-699(November 2005) discloses that As a rule, isotachophoresis is not usedfor the separation of nucleic acids because the mobility ofpolynucleotides in this system does not depend on their size. There isalso disclosed proposed method of agarose gel isotachophoresis of DNAhas been used for the isolation of blood DNA.

Young et al. WO 2009/079028 published 25 Jun. 2009, entitled“Purification and concentration of proteins and DNA from a complexsample using isotachophoresis and a device to perform the purification,”discloses a method of simultaneously co-purifying and concentratingnucleic acid and protein targets into a single volume. The sample isadded to the middle of a device that allows isotachophoresis to occur intwo directions toward both the positive and negative electrodes when avoltage is applied.

BRIEF SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features andaspects of the present invention, nor does it imply that the inventionmust include all features and aspects discussed in this summary.

The present invention comprises, in certain aspects, methods andmaterials useful for fast and efficient extraction, isolation,preconcentration and quantitation of genetic material (e.g., nucleicacids) based on isotachophoresis techniques (ITP).

The invention may be characterized, in certain aspects, as anisotachophoretic method for concentrating a target nucleic acid from asample containing a complex mixture of macromolecules, such as a complexbiological mixture (e.g., blood). The target nucleic acid will typicallybe isolated from other macromolecules, or even small molecules, in aconcentrated zone between the LE and the TE. It may be extracted fromthat zone in a purified form and used for further processing, such asPCR. The method comprises steps including treating the sample, if thetarget is contained in cells, with a cell lysis agent. The cell lysisagent may be mechanical, thermal, electrical or chemical, such as knowncell lysis buffers, suitable for the type of cell being lysed. RBC lysisreagents are commercially available.

The method further involves, if the target nucleic acid is bound toprotein, treating the sample with a release agent to release the targetnucleic acid from the protein. The release agent may be a proteinase,such as proteinase K (EC 3.4.21.64). It can also be used forinactivation of RNAse and DNAse.

The method further comprises applying the treated sample to a samplewell connected to a liquid channel. The sample well and liquid channelmay be separate or integrally formed in a microfluidic chip. The channelmay be an etched channel in a microfluidic device or a capillary tube.In further aspects, the present inventive method comprises contactingthe treated sample from the sample well with a trailing electrolyte(“TE”) having mobility greater than said macromolecules that are nottarget nucleic acid and a mobility less than said target nucleic acid;moving the treated sample from the sample well to the liquid channel,containing a leading electrolyte (“LE”) that has a mobility greater thansaid target nucleic acid, wherein said LE and TE contain electrolytes infree solution, and are at pH between about 4 and 10. The pH may bebetween 6 and 9, or may change during the process and/or may bedifferent between the LE and the TE. The method further comprisesapplying a voltage across the liquid channel containing said treatedsample, LE and TE to cause concentration of said target nucleic acid inan isotachophoresis interface between LE and TE. The contacting of thetreated sample with the TE may involve applying the sample and TEsequentially and/or mixing.

In certain aspects, the present inventive method also comprises methodswhere said applying to a sample well is to a sample well and liquidchannel comprised in a microfluidic device. The separation andconcentration may be achieved in micro-volumes over distances on theorder of millimeters. In certain aspects, the present inventive methodalso comprises the step of adding a polymer sieving agent, such as apolymer, e.g., a block copolymer, a linear polymer of a branched polymerin the liquid channel to change mobility of one or both of targetnucleic acids and molecules not to be isolated. Block copolymers containrepeating oligomers of two or more different polymers.

The invention may further comprise adding an agent for suppressingelectroosmotic flow to the liquid channel. This agent may be selectedfrom the group consisting of polylactams, substituted polyacrylamidederivatives, water-soluble methylhydroxyethyl derivatives of cellulose,polyvinylalcohol, polyvinylpyrrolidones and polyethyleneglycols. Thepolylactam may be polyvinylpyrrolidone.

In certain aspects, the present inventive method also comprises use of adevice where the liquid channel divides at channel bifurcations todistribute various contents of said complex mixture to various channels.An example of this is illustrated in FIG. 13. Mixing may also be carriedout where the sample is mixed with the TE. Mixing may be done off-chip,or on-chip by microfluidic means, e.g., as described in U.S. Pat. No.6,935,772, issued Aug. 30, 2005.

In certain aspects, the present inventive method also comprises anisotachophoretic method for concentrating small RNA from a samplecontaining a mixture of macromolecules and longer RNA. Small RNA may beregarded as RNA smaller than mRNA or tRNA. Major types of small RNAmolecules are small nuclear RNA, small nuclear RNA, micro RNA (miRNA)and short interfering RNA (siRNA). Both miRNA and siRNA are about 20-25nucleotides long. In this method, if the sample contains cells, onetreats the sample with a cell lysis agent, to obtain a treated sample.The method further comprises applying the treated sample from theprevious step to a sample well connected to a liquid channel; contactingthe treated sample from the sample well with a trailing electrolyte(“TE”) having mobility less than said small RNA; moving the treatedsample from the sample well to a liquid channel with a leadingelectrolyte (“LE”) that has a mobility greater than said small RNA,wherein (e) said LE and TE contain electrolytes in free solution, andare at a pH which causes effective mobility of proteins to be differentfrom that of the small RNA; adding a sieving agent to the LE; andapplying a voltage across the liquid channel containing said treatedsample, LE and TE to cause concentration of the small RNA in anisotachophoresis interface between LE and TE.

The method may further comprise the step of treating the sample with anRNAse inhibitor. RNAse may be inhibited by an enzyme that degradesproteins, or by placental protein, antibodies, or commercial productssuch as RNAse inhibitor from Thermo Scientific. The method may alsoemploy a sieving agent, which may be a polymer, including a blockcopolymer of ethylene oxide and propylene oxide. The method may furthercomprise the step of treating the sample with one or more enzymesselected from the group consisting of DNAse and proteinase, which may beproteinase K. In addition, a protein denaturing agent may be added tothe liquid channel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is graphical representation of a buffer system and electrolytesfor use in focusing double stranded DNA but not single stranded DNA. Yaxis is effective mobility μ_(eff) 10⁹ [m² V⁻¹ s⁻¹] plotted versus pH,in acetic acid LE and glycine TE. At pH 10.5, it can be seen that dsDNAfocuses but ssDNA does not.

FIG. 2 is a schematic representation of the ITP-based nucleic acidpurification from a complex biological sample showing a complex sample(2A0, sample beginning to separate as V is applied (2B), andconcentrated sample between LE and TE (2C).

FIG. 3 is a schematic of a design of a microfluidic chip used to performITP-based purification of nucleic acids.

FIG. 4A is an illustration of focused DNA in a microchannel; FIG. 4B isa graph showing results of ITP based nucleic acid purification fromhuman blood (FIG. 4B); focused pg of DNA for different sections of aseparation channel are shown.

FIG. 5A shows a graph and FIG. B a diagram illustrating localization andextraction of ITP purified nucleic acids.

FIG. 6 is a graph showing real time PCR amplification of ITP purifiedDNA from human blood.

FIG. 7A-C is schematic of microchip and protocol for loading PluronicF-127 and extracting, isolating, concentrating and quantitating smallRNAs using ITP. 7A shows filling of the microchannel with sieving agentand LE, at 4° C.; 7B shows addition of small RNAs and TE at +10 min,with temp. 22° C.; and 7C shows ITP focusing of small RNA in the channelbetween the West and East wells and between the LE and the TWE; RNAshave migrated towards the anode.

FIGS. 8A and B are isotachopherograms of separation in 30% PluronicF-127 of (A) 10-100 bp DNA ladder, and (B) of 50-800 bp DNA ladder.

FIG. 9 is a graph showing set of isotachopherograms of extraction andseparation of 22 nt from 66 nt oligos in 30% Pluronic F-127. Lines arein order of legend.

FIG. 10 is a graph showing set of isotachopherograms of extraction andseparation of 22 nt oligos from a 0.1-2 kb RNA ladder byisotachophoresis in 30% Pluronic F-127. Lines are in order of legend.

FIGS. 11A and B is a reverse-image photograph that showsisotachophoretic purification of 22 nt oligos from oligos (11A) yeastcell lysate (11B).

FIG. 12 shows isotachopherograms of atmospheric CO₂-aided simultaneouspreconcentration and separation of 25 bp DNA ladder from greenfluorescent protein (GFP) and allophycocyanin (ACN) in a singleinterface isotachophoresis experiment. Lines are in order of legend.

FIG. 13 is a schematic diagram of an exemplary dual-counterion ITPsystem which can be used to change ITP conditions in real-time. Twodifferent voltages are applied to two branches.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present description is organized as follows:

-   -   I. Overview, describing principles behind the present methods        and materials;        -   A. Selection of electrolyte systems        -   B. Modifications 1-6;        -   C. Definitions    -   II. Examples        -   Examples 1-5—purification of nucleic acids from complex            samples such as whole blood        -   Examples 6-13—separation of different species of nucleic            acids, especially small RNAs.        -   Examples 14-16—additional modifications, suppression of            electroosmotic flow, CO₂ aided extraction and introduction            of counterions

I. Overview

The embodiments of present invention as described below can be used forfast and efficient extraction, isolation, preconcentration andquantitation of genetic material (e.g., nucleic acids) from complexbiological samples based on isotachophoresis techniques. The selectivityof isotachophoretic focusing is used to concentrate nucleic acids in asharp zone while rejecting proteins and other unwanted compounds frombiological samples. The invention enables single step isolation,extraction, preconcentration and quantitation of nucleic acids fromcomplex biological samples like blood, cell lysates, etc usingisotachophoretic techniques while eliminating the need for one or morepreparative steps like centrifugation or filtration. Thus, the presentITP process has been used to isolate target nucleic acids from a ‘soup’of unprocessed biological samples including blood or blood lysate orcell lysates, etc.

The present methods provide high sensitivity, with a lower use of samplereagents: For example, we can currently focus less than 1 pg/μL ofdsDNA. We use reservoir volumes on the order of tens of μL, and can usethis to perform order 10 experiments. We can selectively extract 22-baseRNA from samples also containing >200-base RNA, >100 by DNA andproteins.

In addition, for simple fluorophores, we have demonstrated million foldpreconcentration. For proteins from a complex cell-free expressionbuffer, we have shown 10.000-fold preconcentration in 3 min. The presentITP zones are self-stabilizing and injection protocols are easilyconducted with two pipetting dispensions (e.g., in straight channels).

The present ITP preconcentration and separation assays take 10's to100's of seconds.

The present methods can be combined with counterflow (for stationaryITP) to process large volumes (e.g., order 100 μl volumes of DNA in 30minutes).

The following examples demonstrate that the technique isolates RNAs withlengths of roughly 22 nt (in the range of miRNAs and siRNAs), and thatits selective isolation rejects 66 nt and larger RNAs. We are able toquantitate small RNAs of only ˜900 cells in ˜5 μL, and we believe thatthe sensitivity can be decreased considerably, allowing small RNAextractions from single cells. The examples also demonstrate theapplication of ITP for sample preparation from biological fluids forfurther analysis including for PCR.

Sample ions focus in a sharp zone at the interface between the trailingand leading electrolytes if their charge is of the same sign (i.e., allare either anionic or cationic) and (a) the effective mobility of thesample ions in the TE is faster than that of the TE ions This is shownby formula (1)|{tilde over (μ)}_(s,TE)|>|{tilde over (μ)}_(TE,TE)|  (1)and (b) the effective mobility of the sample ions in the LE is lowerthan that of the LE ions. This is shown by formula (2):|{tilde over (μ)}_(s,LE)|<|{tilde over (μ)}_(LE,LE)|  (2)

Subsequently, equations (1) and (2) determine focusing inequalities. Theeffective mobilities are defined in equation (3):V={tilde over (μ)}E  (3)

Where V is the average velocity of a species and E is the electric fieldin the corresponding zone. The overbar on the μ indicates the effective(not fully ionized) mobility of a weak electrolyte ion. The firstsubscript in mobility indicates the relevant chemical species (“s” forsample and “TE” for trailing ion), and the second subscript indicatesthe zone. {tilde over (μ)}_(s,TE) is therefore the effective mobility ofsample in the TE zone.

A. Selection of Electrolyte Systems

The present methods essentially involve selection of electrolyte systemsincluding buffer systems and chemical mobility modifying agents,according to the above formulas, and as described in detail below. Theelectrolyte system may comprise a solvent (e.g., water) bufferingcounterions, an LE with selected ionic species, a TE with selected ionicspecies, and possible spacers surface-active compounds, and the like.

According to the present methods, the LE and TE are selected to havewith effective mobilities, as defined above, that are higher (in LE) andlower (in TE) than that of the target nucleic acids. Upon application ofan electric field, nucleic acid molecules focus between TE and LE in asharp concentrated zone. Species with smaller effective mobilities thanthe TE migrate into the channel but lag behind and do not focus. Fasterspecies overspeed the sample zone and also do not focus. As an example,at moderate pH and in free solution, DNA has relatively large magnitude(negative) mobility compared to a vast number of polypeptides, so themobility of the TE effectively determines purification selectivity. Theterm “effective mobility” is equivalent to observable mobility. That isthe ratio of velocity to electric field at the specific conditions. Thisis distinguished from “fully ionized mobility in the infinitely dilutelimit” which is thought of as more of a material property.

In typical ITP, analytes at sufficiently high concentration (and aftersufficient focusing time) segregate into distinct zones characterized bya plateau at steady state. The composition of plateau zones is describedfairly generally by the Alberty (R. A. Alberty, J. Am. Chem. Soc., 1950,72, 2361-2367) and Jovin T. M. Jovin, Biochemistry, 1973, 12, 871-878)functions governing ITP electromigration dynamics. However, analytes atinitially trace concentrations rarely have sufficient time (or channellength) to achieve plateau zones. Such analytes focus into approximatelyGaussian peaks, and this regime is thus called peak mode ITP (S. J.Chen, S. W. Graves and M. L. Lee, J., Microcolumn Separations, 1999, 11,341-345). Khurana and Santiago (T. K. Khurana and J. G. Santiago, Anal.Chem., 2008, 80, 6300-6307) presented a theoretical and experimentalstudy for the electrolyte composition optimization in the peak moderegime. We followed their theoretical guidelines in designing our ITPsystem chemistry. The selection of the electrolyte system is crucialsince this allows us to selectively extract and isolate small NAs from alarge variety of other biomolecules present in the cell lysate.

A key aspect of the present method is selection of TE that is justslower (no more) than the target NA and selection of LE that is justfaster than the target NA so as to ensure high selectivity of extractionof NA from a complex biological sample. As described below, the presentmethods involve selection of pH, mobility conductivity and ionicstrength. The LE and TE may have the same or different pH; the mobility

An example of the present methods of LE, TE and buffer selection isshown in FIG. 1.

FIG. 1 shows a graphical representation, in a plot of pH versuseffective mobility, of a system that selectively focuses either single-or double-stranded DNA. Plotted is a schematic representation of theeffective mobility of dsDNA, ssDNA, LE, and TE as a function of pH. Thevalues of the TE and LE pH are shown along the abscissa. Highlighted isthe range where the LE and TE mobilities bound that of dsDNA but notthat of ssDNA. In this zone, ss DNA has lower mobility than the TE inthe TE zone, so it will not focus. Here, the LE is 2-(N-morpholino)ethanesulfonic acid (MES), and the TE is glycine.

To select a particular LE and TE, one can proceed by dichotomy: First,start with LE and TE that have a large focusing window and focus sampleof interest. Then, select a new TE that cuts the previous window by halfand verify focusing. Then, if sample focuses, repeat; or use a new LEthat cuts the window by half and repeat. This process can be iterateduntil the desired selectivity (e.g., LE and TE with +/−1% of the samplemobility) is reached.

By further adjustments of reagents and effective mobilities, the presentisotachophoresis methods are used for simultaneous extraction of DNA andRNA. The trailing and leading ions are selected such that DNA and RNAboth focus at the TE/LE interface, that is, both DNA and RNA meet thefocusing inequalities. Once the microchannel is primed with the LEsolution, and upon application of an electric field in the microchannel,DNA and RNA will focus and concentrate between TE and LE. The TE and LEare chosen such that species not to be focused (e.g., lysed cellmembrane, proteins, etc.) have their electrophoretic mobility eithersmaller than the trailing ion or larger than the leading ion. The slowerspecies will remain in the TE well while faster species exiting the TEwell overspeeds the nucleic acid zone and proceeds to the LE well. Forshort focus times, a finite quantity of fast species remains in the TEwell. These can be removed completely given longer focusing times (e.g.,via the application of counterflow). In one embodiment, it is possibleto reduce the electroosmotic flow (EOF) by coating the channel walls.Such a coating can enhance the recovery of nucleic acids by reducingtheir adsorption on the walls.

By further adjustment of reagents and effective mobilities, ITP is usedherein to selectively extract RNA and not DNA from the lysed cellsample. In this case, LE and TE mobilities are chosen such that only RNAmeets the focusing inequalities, while DNA migrates through the TE/LEinterface and into the leading well. This scheme assumes that the RNAmobility is less than that of DNA. If conditions are such that RNAmobility is greater than DNA, then buffers can be chosen such that RNAmeets the focusing inequalities, but DNA does not.

For the latter, we need electrolytes with mobility values bounded bythat of RNA and DNA. This may be accomplished by leveraging thesize-dependent mobility of DNA and RNA in free solution. Alternately, wecan use a sieving matrix to affect a strong size dependence on RNA andDNA.

The samples contemplated here will have a variety of molecular species.One species will be concentrated in the interface between LE and TE.

Examples of LE and TE are chloride ions as LE and 6-aminocaprroic acidas a TE; Tris+HCl as an LE and TRIS HEPES as a TE; 6-aminocaproic acidand HCl as LE and 6-aminocaproic acid and caproic acid or Bis-Tris andDihydroxybenzoic Acid as TE; and Tris-HCl as LE and glycine orTRIS-glycine as TE. These are adjusted as to concentration and buffersto provide the effective mobility differentials as described above.Exemplary buffering counterions include: mM BisTris, pH 6.0; mM Tris, pH7.6; 267 mM BisTris and 100 mM NaOH, pH 6.7.

B. Modifications to Selective LE/TE Buffer Components

In addition to the methods taught here for selecting the LE, TE andother components for focusing of the desired nucleic acid species, othersteps can be employed for further enhancements:

(1) A sieving agent may be added in the separation channel to changemobility of the macromolecules not to be concentrated. In one aspect ofthe invention, small RNAs are extracted, isolated, preconcentrated andquantified from complex biological samples using ITP in microchannelsloaded with a high efficiency sieving medium. An example of a sievingmedium is Pluronic F-127. Pluronic F-127 is a triblock copolymerPEO-PPO-PEO, where PPO is poly (propylene oxide) and PEO is poly(ethylene oxide). At temperatures below 15° C. the viscosity of thisamphiphilic copolymer solution is low because PPO is hydrophilic, andPluronic F-127 dissolved in water acts as a free flowing solution. Whenthe temperature is increased, the hydrogen bonds between PPO and waterare broken, leading to a high degree of PPO hydrophobicity so thatmicelles are formed. These spherical micelles with hydrophobic PPO coressurrounded by PEO brushes are packed in a face-centered cubicnanostructure, forming a liquid crystalline phase. It was measured for20% Pluronic F-127 that the viscosity at 4° C. is ˜50 cP compared to avalue of ˜900 cP at 22° C. The mobility of small RNAs in this sievingmatrix decreases with increasing number of nucleotides. We have searchedfor and found an effective mobility for our TE which is between themobility of small RNAs and pre-miRNAs in the sieving matrix, henceseparating these species. These were determined from simulations usingPeakmaster 5.2 for the buffer calculations (including ionic strengthdependence).

Hydrophilic polymers such as linear low-molecular-mass polyacrylamide orlow molecular-weight poly(ethylene oxide) (PEO) are suitable sievingpolymers.

Block copolymers comprise two or more homopolymer subunits linked bycovalent bonds. The union of the homopolymer subunits may require anintermediate non-repeating subunit, known as a junction block. Blockcopolymers with two or three distinct blocks are called diblockcopolymers and triblock copolymers, respectively. Copolymers may also bedescribed in terms of the existence of or arrangement of branches in thepolymer structure. Linear copolymers consist of a single main chainwhereas branched copolymers consist of a single main chain with one ormore polymeric side chains. A number of different monomers are known foruse in preparing block copolymers, including isoprene and styrene.

A sieving matrix would also allow the selective focusing of DNA with anyrange of lengths

(2) Electroosmotic flow suppression/microchannel treatment is effectedby addition of a chemical agent to enhance selectivity. Examples ofagents used to suppress electroosmotic flow include polylactams, such aspolyvinylpyrrolidone, substituted polyacrylamide derivatives,water-soluble methylhydroxyethyl derivatives of cellulose,polyvinylalcohol, polyvinylpyrrolidones and polyethyleneglycols andnon-ionic detergents like Triton X-100. In one embodiment, LE may be ofhigh mobility, thus increasing ITP velocity and decreasing the detectiontime.

The microfluidic channels may be treated for electroosmotic flowsuppression or for other beneficial flow modifying effects. Themicrochannels may be of a non-conducting material like silicate orborosilicate. The microchannels may be pretreated with one or moreagents including silanizing agents, alcohols, acids and water. Themicrochannels may be present in a simple cross geometry, that is, theymay be branched at one end, as illustrated, or may be in a combconfiguration, with several branches.

Polyvinylpyrrolidone, formula (C₆H₉NO)_(n), CAS 9003-39-8, and may bethe lower molecular weight form, about 40,000 or the higher form, about360,000.

(3) Sample disruptive or degradative agents are used to treat a complexbiological sample. These agents are those for lysing cells in a wholeblood or other cell-containing sample, degrading proteins or lipids in asample, or the like. Examples of lysis agents include detergents andsurfactants, preferably non-ionic surfactants. Examples of non-ionicsurfactants include Triton-X-100 and Igepal CA-630. In one embodiment,the lysis buffer may not include chaotropic agents. In one embodiment, anuclease (DNAse or RNAse) or a nuclease inhibitor may be added to thelysis buffer to ensure selectivity of the nucleic acid being purified(DNA or RNA). In another embodiment, a protease enzyme like Proteinase Kis added to the lysis buffer.

A proteinase, also known as a protease, is an enzyme that breaks downproteins by hydrolyzing peptide bonds. Protease enzymes (like ProteinaseK) may be used to degrade proteins which potentially inhibit PCR intoshort polypeptides. However, proteinase K is itself a PCR inhibitor. Wecompensate for this by operating with an ITP chemistry where proteinaseK (pI=8.9) is positively charged and is, therefore, kept away from thesample zone as it electromigrates in the opposite direction (enters andremains in the TE reservoir). The combination of proteinase K andITP-based purification effectively removes PCR inhibiting species andother polypeptides from the biological sample. During purification, thebiological sample is hydrodynamically injected between LE and TE. Uponapplication of an electric field, nucleic acids focus and migratetowards the anode in a sharp concentrated zone. When the focused nucleicacids enter the anode reservoir, they are pipetted out (in a very smallvolume up to 5 μl), added to a PCR mix and real time PCR is performed.

In one embodiment, a single mixture of lysing agent and TE is added tothe sample and ITP is performed for separation and concentration ofnucleic acids. The single mixture serves two functions: lysing cells inthe sample as well as being the trailing electrolyte for focusing thenucleic acids in the sample.

The location of the focused nucleic acid ITP zone may be tracked bymonitoring ionic current or by fluorescence visualization. Fluorescencedyes like SYBR Green are used to intercalate between nucleic acids tofacilitate detection of the purified nucleic acids. Invertedepifluorescence microscopes may be used to detect the isolated andpurified nucleic acids. Images are captured on a CCD camera. Standardand ITP-focused images are corrected with the background and their ratioobtained. An integrated normalized intensity of the nucleic acid iscalculated and then converted to nucleic acid mass. Calculations may bedone using standard software packages like MATLAB (The Mathworks,Natick, Mass.).

(4) Counterions may be used to tune the mobilities of the LE and TE.

Selective focusing of nucleic acids may be enhanced with multiplecounterions to independently tune the effective mobilities of the TE andLE. In conventional ITP, the effective mobility of the TE is coupled tothat of the LE because the pH of the LE has a strong influence on the pHof the TE and sample regions. pH largely determines the effectivemobility of the species. In embodiments of the present invention, therelation between the pH of the TE and the pH of the LE is changedsubstantially by adding a second counterion to the LE to modify the pHof the TE (while the pH of the LE remains approximately constant). Bylargely independently tuning the effective mobilities of the LE and theTE, very precise, fast, and efficient separation of the sample ispossible. Since pH largely determines the effective mobility of aspecies, this method uncouples the effective mobilities of the twoelectrolytes.

A dual ion process described here can be used in a real-time adjustmentof chemistry performed with a microchip with two LE wells as describedin Example 16 below.

(5) Carbon dioxide e.g., from the atmosphere, may be used to achieve ITPwhich simultaneously preconcentrates and separates analyte species.Since the wells are open to atmosphere, carbon dioxide continuallyreacts and enters the channel. CO₂ reacts with water to form a carbonatezone. A carbamate zone forms directly from the reaction of dissolved CO₂with primary and secondary amines present in the solution. Withappropriate LE and TE chemistries, these carbonate and carbamate zonesfocus and also create electric field gradient region between the LE andTE boundaries. The analytes focus in this gradient region based on theirelectrophoretic mobility and we achieve simultaneous focusing andseparation of these analytes. Analytes separated include nucleic acidsfrom a complex biological sample or one or more proteins from a complexbiological sample.

(6) Counterflow of the bulk liquid may be used to increase the focusingtime and the amount of focused material. The counterflow (of thesolvent) is applied in the opposite direction of ITP electromigration.This counter flow can be achieved using either a pressure gradient tocreate pressure-driven flow, or by taking advantage of theelectroosmotic flow (e.g., as in the case of the electrophoresis ofanions in a channel with a negative wall surface charge). The counterflow of the background liquid in the direction opposite of theelectromigration will slow down the sample and therefore increase itsresidence time within the capillary. Increased residence time allows forpreconcentration of trace analytes from relatively large volumes intosmall, recoverable sample volumes. In addition to long term stacking,the process also allows larger separation times of ionic species byisotachophoresis.

C. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. Generally, nomenclatures utilized inconnection with, and techniques of, cell and molecular biology andchemistry are those well known and commonly used in the art. Certainexperimental techniques, not specifically defined, are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. For purposes of theclarity, following terms are defined below.

The terms “isotachophoresis,” ITP, or isotachophoretic, is used in itsconventional sense, and refers to a nonlinear electrophoretic techniqueused in the separation of a variety of ionic compounds, as described indetail below. Unlike “linear” zone electrophoresis in which separatingsolute bands continually spread by diffusion or dispersion, ITP formsself-sharpening, adjacent zones of substantially pure solute. Sample isusually introduced between the leading electrolyte (LE, containingleading ion) and the terminating electrolyte (TE, containing terminatingion) where the leading ion, the terminating ion and the samplecomponents must have the same charge polarity, and the sample ions musthave lower electrophoretic mobilities than the leading ion but largerthan the terminating ion. After application of a fixed electric current,sample components move forward behind the leading ion and in front ofthe terminating ion and form discrete, contiguous zones in order oftheir electrophoretic mobilities. Then, following a brief transientperiod where the discrete solute zones are formed, this ITP “stack”assumes a fixed concentration profile with a constant velocity moving inthe direction of the leader. ITP differs from capillary electrophoresisin that it uses a discontinuous buffer system as described above. Also,capillary electrophoresis is characterized by the use of high voltages,which may generate electroosmotic and electrophoretic flow of buffersolutions and ionic species, respectively, within the capillary. Theproperties of the separation and the ensuing electropherogram havecharacteristics resembling a cross between traditional polyacrylamidegel electrophoresis (PAGE) and modern high performance liquidchromatography (HPLC).

The term “nucleic acids” or “polynucleic acids” refers to polymeric oroligomeric strands of DNA or RNA. RNAs include small RNAs such assiRNAs, miRNAs and piRNAs. siRNAs are generated from double-strandedRNAs (dsRNAs) which are cleaved to ˜21-25 nucleotides (nt) by Dicer, anendonuclease belonging to the RNaseIII family serving as a molecularruler. By contrast, miRNAs are derived in a two-step process. Theprimary precursors of miRNAs (pri-miRNAs) are encoded in the genome,having lengths of several hundred to thousands of nucleotides. Inanimals, the pri-miRNAs are then processed to ˜70 nt pre-miRNAs whichare transported into the cytoplasm, where the pre-miRNAs are cleaved toproduce mature ˜21-25 nt miRNA-iRNA* duplexes (where miRNA is theantisense, or guide, strand, and miRNA* is the sense, or passenger,strand). Biogenesis of the third class, piRNAs which are single-strandedRNAs (ssRNAs) and ˜24-31 nt long, is distinct from that of siRNAs andmiRNAs and does not involve dsRNA precursors.

The term “microfluidic device” is used in its conventional sense torefer to a device which is typically on a chip, for carrying out fluidmanipulation, “on chip.” The device contains a sample well andmicrochannels is that the depth dimensions of etched channels (typically10-20 μm deep). A sample is introduced into the microchannel systemusing various electrokinetic—or pressure—injection methods. U.S. Pat.No. 6,695,009, whose contents are incorporated by reference to theextent necessary to understand the present invention, shows one priorart approach to sample stacking. Further description of an exemplarymicrofluidic device may be found in Santiago et al. US 2006/0042948 A1,entitled “Microfluidic Electrophoresis Chip Having Flow-RetardingStructure.” A flow retarding structure is not required for presentpurposes. The present microchannels may also be capillary tubes, whichare generally about ½ mm in internal diameter. Channel areas may be,e.g., 1000-5000 μm².

The term “electroosmotic flow” refers to the motion of liquid induced byan applied potential across a porous material, capillary tube,microchannel, or other fluid conduit. Because electroosmotic velocitiescan be independent of conduit size, whereas flow due to pressuregradients is much more significant with large conduits, electroosmoticflow is most important when the fluid conduit is small. Electroosmoticflow is an essential component in certain chemical separationtechniques, notably capillary electrophoresis.

II. Examples Example 1 Purification of Nucleic Acids from Whole BloodUsing Isotachophoresis

As shown in FIG. 2A-C, the present principles of nucleic acidpurification from complex samples involve the use of an LE (circles 206)and TE (squares 204) in a microchannel 202. The sample initiallycomprises a plug of mixed components, shown as DNA (double lines 210),RNA (curved lines 208) (collectively “NA”) and proteins and othercontent (stars 212). In the middle panel FIG. 2B, the initial separationof nucleic acids by moving in to the LE is shown. In the bottom panelFIG. 2C, the NA are shown between the LE and TE, while other componentsremain in the TE. That is, LE and TE are selected with mobilities,respectively, larger and smaller than NA. The TE needs to have largermobility than proteins (and other contents) present in blood lysate. Weinject a finite plug of lysate between TE and LE. Upon application of anelectric field, NA focus between LE and TE, while proteins cannot focusas they travel slower than the ITP interface. After sufficient time, theITP zone contains only pure NA extracted from the lysate.

The design of the microchip used in these experiments is shown in FIG.3. FIG. 3 shows a microfluidic chip which has four wells, with twochannels terminating in wells 1 and 3 and 2 and 4, respectively. Thechannel between wells 1 and 3 provides an injection channel. Thiscrosses a purification channel between wells 2 and 4 (the extractionwell). Sample is injected from reservoir 3 by applying a vacuum at 2.Typically all four channels and reservoirs were filled with LE.Reservoir 3 is emptied with a vacuum and as little as 1 μL of sample isadded into that reservoir. Then a vacuum is applied to reservoir 2 tofill the injection channel with the sample. We carefully removed thesample remaining in reservoir 3 and then rinsed and replaced it with TE.We then immediately applied an electric field between 3 and 4 (0-3000 V)with a sourcemeter (model 2410, Keithley, Ohio) to carry out thepurification. We used the current signal to locate the ITP interface inthe channel, as described further below in connection with Example 3.

Microchip Preparation: We performed on-chip experiments in a microchipwith 90 μm wide by 20 μm deep borosilicate microchannels in a simplecross geometry (FIG. 7) (model NS12A, Caliper Life Sciences, CA).

We treated the channels with the silanizing agent Sigmacote (Sigma, Mo.)as follows. We first rinsed the channel 10 min with a 1:1methanol/hydrochloric acid solution, followed by 10 min of concentratedsulfuric acid. We then rinsed the channels with deionized water for 2min or more and dried them thoroughly with a vacuum. Next, we appliedthe silanizing solution for about 10 min. We then rinsed the channelswith hexane and deionized water. To avoid cross contamination, we rinsedthe chip between each experiment as follows: 2 min with a 1:10 (v/v)household bleach solution (Clorox, CA), 2 min with deionized water, and2 min with leading electrolyte buffer (see below).

Sample Lysis: Blood samples from a healthy donor were collected inheparin tubes and stored in 2 mL aliquots at −80° C. Before each set ofexperiments, we thawed one blood aliquot and prepared a stock of lysisbuffer containing 1% Triton X-100 (Sigma, Mo.) in 50 mM Trishydrochloride at pH) 8.2. We diluted 10 μL of whole blood and 4 μL ofproteinase K (RNA grade, Invitrogen, CA) in 86 μL of lysis buffer. Wethen incubated the lysate 10 min at 56° C. in a hot bath. In the case ofthe second control in FIG. 4 (third bar in the plot), we added 4 U ofdeoxyribonuclease I (DNase I, amplification grade, Invitrogen, CA) tothe lysate and incubated for 15 min at room temperature prior toproteinase K treatment. To quantify the ITP extraction efficiency, wediluted a commercial standard solution of k-DNA (0.333 mg mL-1,Invitrogen, CA) in lysis buffer and used this as a standard sample. Allsolutions were prepared with DNase/RNase free deionized water (Gibco,CA).

Isotachophoresis-Based Purification: Leading (LE) and trailingelectrolytes (TE) were, respectively, 50 mM Tris titrated withhydrochloric acid to pH) 8.2 and 50 mM Tris titrated with HEPES topH=7.8. LE and TE each contained 1×SYBR Green I (Invitrogen, CA) forfluorescence visualization and on-chip DNA quantitation. We obtained thebest results adding also 0.1% Triton X-100 to reduce electroosmotic flowand protein adsorption (in conjunction with silanization treatment). Foreach experiment, we first filled all four channels and reservoirs withLE. We emptied reservoir 3 with a vacuum and pipetted 1 μL of lysateinto that reservoir. We then applied a vacuum to reservoir 2 to fill theinjection channel with lysate (˜25 mL). We carefully removed the lysateremaining in reservoir 3 and then rinsed and replaced it with TE. Wethen immediately applied an electric field between 3 and 4 (500 V) witha sourcemeter (model 2410, Keithley, Ohio) to carry out thepurification. We used the current signal to locate the ITP interface inthe channel (see below, Example 4). We used this same injection protocolfor the extraction efficiency quantitations performed with k-DNA.

Visualization: We performed on-chip visualization on an invertedepifluorescent microscope equipped with a 4×(Plan APO, N.A.=0.2, Nikon,Japan) or a 10× objective (Plan APO, N.A.=0.45); a mercury light source(Ushio, Japan); a filter cube (exciter/emitter 485/535 nm, Omega, VT);and a 0.6× demagnification lens (model RD060-CMT, DiagnosticInstruments, MI). We acquired images with a CCD camera (Cascade 512F,Roper Scientific).

On-Chip Quantitation: We quantified the amount of DNA extracted fromwhole blood by first calibrating our fluorescence measurement. For thecalibration, we used a control solution of genomic DNA purified fromblood with the DNeasy blood and tissue purification kit (QIAGEN, CA). Wemeasured its DNA concentration with a Nanodrop 1000 spectrophotometer(ThermoScientific, MA) and prepared a 1.42 μg mL-1 standard solutionstained with 1×SYBR Green I in LE. We acquired images of the fluorescentprofile of this standard filling the purification channel (but withoutperforming ITP). Using these images, we were able to relate peak areasto DNA mass in the ITP experiments. Raw data I for a DNA ITP zone from aDNA purification experiment from blood lysate can be imaged as a band.The fluorescence profile I_(std) of the channel filled with the standardDNA solution (here 1.42 μg.mL⁻¹ of human genomic DNA) can also bedetermined. Also, the background fluorescence I_(bgd), where the channelis filled with deionized water. We correct both ITP-focused DNA andstandard images with the background, and then take their ratio. Thisyields an image where the value of each pixel is in units of standard.Summing over all pixel yields the integrated, normalized intensity F interms of these standard units. To convert this number to DNA mass, wetake its product with the standard concentration and the volume of eachpixel (pixel area times the channel depth, assuming an approximatelyrectangular channel cross section). We performed all calculations usingMATLAB (The Mathworks, MA). We also performed this calibration with asolution of λ-DNA.

The following formula was used:

$F = {\Sigma^{{all}\mspace{14mu}{pixels}}\frac{I - {{Ib}\;{gd}}}{{Istd} - {Ibgd}}}$

Where I is intensity and a standard solution is compared againstbackground.

-   -   Focused DNA mass=F×p_(std)×V_(pixel)    -   P_(std)=concentration of standard solution (pg/nL)    -   Vpixel=pixel volume    -   =pixel width×pixel height×channel depth.

Referring again to FIG. 4, the bars show mean values of NA mass purifiedfrom blood lysate, as calculated from the fluorescence intensity profiledescribed above. Purifications of DNA from about 2.5 mL of whole blood(25 mL of blood lysate) provided results from three sets of experiments:purified blood lysate, blood lysate treated with proteinase K, and bloodlysate treated first with DNase and then proteinase K. We used SYBRGreen I fluorescence measurements to estimate the amount of DNArecovered in the ITP zone (see above, Example 1). The amount of DNArecovered without proteinase K is negligible (on the same order as thenegative control). The mass of focused DNA is significant whenperforming the assay on a lysate initially treated with proteinase K.The third set of results shows ITP purification of a lysate treated withDNase prior to proteinase K treatment. This control case shows low DNArecovery, as expected. We hypothesize that DNA binding proteins (inparticular histones) significantly reduce the electrophoretic mobilityof DNA by increasing the hydrodynamic Stokes' drag of the complex. Ifthe mobility of the DNA-protein complex is smaller than the mobility ofthe TE, DNA does not focus and cannot be purified. Proteinase K releasesDNA from binding proteins allowing focusing and purification. Together,these experiments show that our fluorescence signal is due to focusedDNA from the lysate samples and that the extraction process is suitablyrepeatable. We show an image of ITP-focused DNA zone above the bargraph. The amount of focused DNA is 44.2±6.2 pg. We injected 2.5 mL ofblood, which contains between 65 and 162 pg of DNA. A nanoliter of bloodfrom a healthy human contains 4-10 white blood cells, and each humandiploid cell contains about 6.6 pg of DNA so that our purificationefficiency for a blood sample ranges between 30% and 70%, which competeswith both batch and microchip-based SPE methods. Uncertainty barsrepresent uncertainty in measured mass of DNA with a 95% confidenceinterval. The three sets are results from N=2, 9, and 2 repetitions,respectively.

Example 2 Characterization of Extraction Efficiency of Purified DNAProduced

In purification procedure for DNA, there are at least two extractionefficiencies of interest: the fraction of DNA purified and focused viaITP from the amount injected into the channel and the amount of DNAextracted from the chip and delivered to PCR versus the amount of DNA inthe lysate dispensed into the chip. To characterize the former, weapplied our technique to a solution of λ-DNA of known concentration. Weinjected 10 pg of λ-DNA on-chip and performed the ITP purification asdescribed in Example 1. We determined the amount of focused DNA byquantifying SYBR Green I fluorescence and comparing it to the standard.We measured the mean fraction of extracted DNA (v. amount injected intothe channel) as 1.03±0.06 (N=3). This approximately complete extractionis at least as good as traditional extraction methods (e.g., QIAGENkits) and state-of-the-art microsolid phase extraction devices 6.Weestimate that we extract out of the chip (and deliver to the PCR)approximately all of the DNA which we focus on the chip.

Example 3 Localization and Extraction of ITP-purified DNA

We track the location of the focused NA ITP zone by either monitoringionic current or by fluorescence visualization. FIG. 5 shows a measuredcurrent trace obtained from a constant voltage ITP extraction experimentwhere the sample is a solution containing 7 ng.; L-1 of lambda λ-DNA. Weacquired the current trace by interfacing the sourcemeter with MATLABusing a GPIB card (National Instruments, TX). The current decreasesmonotonically as the ITP interface advances within the channel, as therelatively low conductivity TE replaces the high conductivity LE. At themoment where the current reaches a plateau (here near t=260 s), thepurification channel is entirely filled with TE and the ITP interfacehas reached the anode reservoir. Above the current plot (5A), is anactual image of focused DNA in the microchannel (with a superposedschematic of walls). FIG. 5B shows drawings of actual fluorescenceimages corresponding to the same experiment. Image 1 shows the focusedDNA approaching the reservoir. Image 2 shows an image of the samelocation just after the interface enters the reservoir. Image 3 showsthe reservoir about 20 s later, where the purified NA has migrated intothe reservoir. These three instances in time are highlighted in thecurrent plot. Either or both current monitoring and fluorescencevisualization can be used to track the position of the NA during thepurification process.

Example 4 Off-Chip PCR of Purified DNA

We tracked the position of the focused zone in the channel by directlyvisualizing the focused species or by monitoring current transients (seeabove, Example 3). After the ITP interface exited the purificationchannel, we collected the liquid from reservoir 4 (˜2 μL) with astandard pipettor into a PCR tube containing 5 μL of 2× Fast SYBR GreenI master mix (Applied Biosystems, CA) and 0.1 μM primers (Invitrogen,CA) targeting a 201 bp fragment of the BRCA2 gene and filled the rest ofthe reaction tube with deionized water up to 10 μL.

The primer sequences for the BRCA2 201 bp fragment amplification are thefollowing:

Forward primer: CAC CTT GTG ATG TTA GTT TGG A (SEQ ID NO: 1) Reverseprimer: TGG AAA AGA CTT GCT TGG TAC T (SEQ ID NO: 2)

The procedure may be summarized as follows: At t₀, the lysate isinjected and is concentrated near the TE well, interfacing with the LE.As time passes, the focused NA move in to the LE and through thepurification channel, becoming further concentrated, until at the endtime they are a narrow band at the LE end, where they are collected in asample tube and diluted up to 10 μL. The material is pipetted from thewell and processed further, in this case it can be successfullyamplified by PCR, meaning that the sample does not contain materialsthat interfere with PCR, as by blocking enzymatic activity or DNAhybridization. All PCR reactions were prepared in a UV-sterilized fumehood to avoid contamination and allow sensitive amplification withoutfalse positives. We performed off-chip real-time PCR on an ABI 7500 Fastthermocycler with the following thermal profile: 20 s initial hold at95° C. and 40 cycles composed of 3s denaturation at 95° C. followed by30 s annealing and extension at 60° C. We performed post-PCRdissociation curve analyses on the same instrument.

Example 5 Real-time PCR Amplification of ITP-purified DNA from HumanBlood

FIG. 6 shows the results of real time PCR amplification of ITP purifiedDNA from human blood. The amplification curve shows repeatableamplification of the extraction product (here four repetitions,threshold cycle C_(t)=30.9±0.4). Negative controls and PCR fromequivalent amount of lysate showed no amplification after 40 cycles. We,therefore, successfully purified DNA from whole blood to obtainPCR-ready NA in a PCR-friendly buffer. The inset shows the post-PCRdissociation curve (derivative of SYBR Green I fluorescence). Themelting temperature of the PCR product equals that of the positivecontrol (T_(m)=75.5° C., see dissociation curves on the inset of FIG.6). The dashed line corresponds to melting curves of negative controls.The experiment showed that we successfully and reproducibly purified DNAfrom whole blood and recovered genomic DNA free of PCR inhibitors.

Example 6 Selective Extraction, of Small RNAs from Cell Lysate UsingOn-chip Isotachophoresis and Sieving Agent

Chemicals and reagents: 6-aminocaproic acid, caproic acid, Tris,Bis-Tris, HEPES, Pluronic F-127 (registered trademark of BASF), IgepalCA-630, and sodium chloride were purchased from Sigma-Aldrich (St.Louis, Mo.). HCl and magnesium chloride hexahydrate were from EMDChemicals (Gibbstown, N.J.). 22 nt and 66 nt oligos (both mixed base A,C, G) were from Integrated DNA Technologies (Coralville, Iowa). SYBRGreen I and II; 50 base pairs (bp), 123 bp, and 250 bp DNA ladders;0.1-2 kb RNA ladder; 293A cell line; streptavidin (Alexa Fluor 488conjugate); albumin from bovine serum (BSA, Alexa Fluor 488 conjugate);and ultrapure DNase/RNase-free distilled water were purchased fromInvitrogen (Carlsbad, Calif.). The 10 bp DNA ladder was from Promega(Madison, Wis.). We purchased embryonic kidney (293) total RNA, RNaseinhibitor, and RNaseZap wipes from Ambion (Austin, Tex.).

Cell lysate preparation: Cell lysing was preformed off-chip according toa standard procedure. We followed the first three steps of the RNeasyMini Kit supplementary protocol from QIAGEN (Hilden, Germany). First,3×10⁶ 293A cells were pelleted by centrifuging for 5 min at 300g and thesupernatant was removed by aspiration. Second, the cells wereresuspended in 175 μL of precooled (4° C.) lysis buffer and incubatedfor 5 min on ice. The lysis buffer contained 50 mM Tris and 30 mM HCl(pH 8), 140 mM NaCl, 1.5 mM MgCl2, 0.5% (v/v) Igepal CA-630 which is anonionic detergent, and just before use, we added 1 U μl-1 RNaseinhibitor. Third, we centrifuged the cell lysate at 4° C. for 2 min at300g and recovered the supernatant which contained the cell lysate.

Electrolyte composition: We experimented with and evaluated theperformance of over 100 ITP buffer combinations (in free solution andwith a sieving matrix) and several labeling techniques. Here, wedescribe our experiments with the best performing chemistry. We used anLE with a high mobility, 140 mM 6-aminocaproic acid and 100 mM HCl (pH4). This LE has a high mobility compared to the TE, increasing the ITPvelocity and therewith decreasing the detection time. The TE was 10 mM6-aminocaproic acid and 50 mM caproic acid (pH 4), which mobility allowsthe separation of small RNAs and premiRNAs. The electrolyte system hasbeen designed to be at pH 4, at which value most proteins contained inthe cell lysate are positively charged since they have a pI>4.26 Thus,most proteins do not migrate through the microchannel and remain in thesample reservoir. We verified the latter with a set of preliminaryexperiments using streptavidin and albumin from bovine serum (eachlabeled with Alexa Fluor 488).

The pH values of the electrolytes were measured with Corning Pinnacle542 pH/conductivity meter from Nova Analytics (Woburn, Mass.). Allbuffers were prepared with distilled, ultrapure, 0.1 micron filtered,RNase and DNase free water, and contained the intercalating dye 1×SYBRGreen I for (dsDNA) or II (for RNA). For the experiments with lysed 293Acells we worked at concentrations of 100×SYBR Green I and II. Theseintercalating dyes are positively charged when they are free insolution, and so conveniently do not focus in our anionic ITPexperiments. SYBR Green I and II have a much lower fluorescence quantumyield when free in solution than when complexed with either dsDNA orRNA.27 These dyes are used effectively in a variety of analytical anddiagnostic applications for the detection of nucleic acids.

Sieving matrix: For the separation of 22 nt from 66 nt oligos we chosePluronic F-127 at a concentration of 30% (w/v), which has demonstratedto be highly effective for the sieving of oligos with lengths of 8-32nt.17,29 The triblock copolymer was dissolved in the LE and placed inthe refrigerator (4° C.) for a few days prior use to ensure completedissolution of the powder.

Imaging: For imaging we used the inverted epifluorescent microscope IX70from Olympus (Hauppauge, N.Y.), equipped with a 100 W mercury lamp, a10× UPlanF1 objective (NA 0.3), and XF115-2 filtercube (455-490 nmexcitation, 510 nm emission, 505 nm cutoff dichroic) from Omega Optical(Brattleboro, Vt.). Images were captured with a MicroMax 1300 CCD cameracontrolled with WinView32, both from Princeton Instruments (Trenton,N.J.). For image analysis we used ImageJ from the National Institutes ofHealth and MATLAB from MathWorks (Natick, Mass.).

On-chip isotachophoresis experiments: We performed all experiments onNS-95 glass microchips from Caliper (Mountain View, Calif.), having asimple-cross geometry with wet-etched 12 μm deep by 34 μm wide channels.FIG. 7 shows a schematic of chip and experimental steps.

FIG. 7A shows filling of the microchannel with matrix; FIG. 7B shows thepositions of the reagents after 10 min at about 22 deg. C. FIG. 7C showsITP focusing of small RNAs between the LE and the TE as the materialmoves towards the anode. Overall, there is illustrated a schematic ofthe microchip and protocol for loading of the high efficiency sievingmedium and extracting, isolating, preconcentrating and quantitatingsmall RNAs using ITP. The microchip is first placed in the freezer for aperiod of time. Then, the north and south reservoirs are filled with LE,and the west well with the sieving medium. (a) For filling themicrochannels with solutions, a vacuum is applied at the east reservoir,and immediately thereafter the west well is cleaned and refilled withLE. (b) After a warm-up period, the sieving medium transitions into ahigh-viscosity solution, and the west reservoir is filled with TE andsmall RNAs. (c) The separation and focusing of small RNAs is initiatedby applying the anode in the east well and the cathode in the westreservoir. The ITP zone travels at constant velocity v downstream(iso-tacho). Small RNAs are detected just to the left of theintersection.

The separation channel from the west well to the cross has a length of27.5 mm. For loading the separation channel with Pluronic F-127 the chipwas placed in the freezer (−10° C.) for 10 min. We then loaded the northand south reservoirs with LE buffer and the west well with refrigeratedPluronic F-127 solution (T≈4° C.), and applied a vacuum at the eastreservoir for 2 min (FIG. 7A). Since the resistance of the north andsouth channels is much lower than that of the west channel, the PluronicF-127 sieving matrix effectively only fills the west channel. The eastchannel is filled primarily with LE. (Some small amount of PluronicF-127 is drawn into the east as thin stream, but this Pluronic F-127“fiber” was negligibly small and often not detectable.) Aftervacuum-assisted channel loading the remaining sieving solution wasremoved from the west well by aspiration with a pipette tip and vacuumline, and refilled with LE to guarantee repeatable results. We let thesystem warm up to room temperature for 10 min, during which periodPluronic F-127 formed a crystalline phase. After the warm-up period, weperformed a pre-run at electric field strength of 200 V cm⁻¹ tostabilize the sieving matrix. For the application of the electric fieldwe used the LabVIEW-controlled high-voltage power supply MicrofluidicTool Kit from Micralyne (Edmonton, AB, Canada) and placed platinum wiresin the east and west reservoirs. Then, the LE in the west well wasreplaced by a mixture of TE and sample (FIG. 7B), and the electric fieldof 400 V cm⁻¹ was applied in east-west direction. As shown in FIG. 7C,the sample zone is focused between the LE and TE and travels at constantvelocity downstream, increasing in concentration and amount of samplewith time as the ITP is in peak mode. Fluorescence signals werecollected at the end of the separation channel just to the left of thecross. After each experiment Pluronic F-127 was removed from theseparation channel by refrigerating the device and applying a vacuum atthe west reservoir for a few minutes. Then, we cleaned the device with0.1 M KOH for 15 min and thereafter with distilled, ultrapure water.Additionally, when working with 293 total RNA or 293A cell lysate, wecleaned all surfaces with RNaseZap wipes to ensure an RNase-freeenvironment.

Transient isotachophoresis: The sieving efficiency of Pluronic F-127 wasinvestigated and demonstrated using transient ITP (tITP). tITP firstpreconcentrates samples in a preliminary ITP step. The ITP step is theninterrupted by injecting LE ions behind the TE region, and thisinitiates electrophoretic separation of analytes. We used a tITPprotocol similar to that described by Bharadwaj et al. (R. Bharadwaj, D.E. Huber, T. Khurana and J. G. Santiago, in Handbook of Capillary andMicrochip Electrophoresis and 105 Associated Microtechniques, ed. J. P.Landers, CRC Press, 3^(rd) edn., 2008, ch. 38, pp. 1085-1120). To loadthe sieving matrix we followed the same procedure as described in FIG.7, but for tITP experiments the TE was 10 mM Bis-Tris and 50 mM HEPES(pH 5.4). This TE has a lower mobility than that for the other ITPexperiments, hence also focusing relatively low mobility species such aslong nucleic acids, for example. After a few seconds of ITPpreconcentration we turned off the electric field, quickly replaced thesolution in the west reservoir with LE, and applied an electric field of1200 V cm⁻¹. This way, the ITP mode is terminated by injecting LE intothe separation channel. Leading ions overtake first trailing ions andthen sample ions, thus initiating a separation of sample species bycapillary electrophoresis.

Example 7 Selection of the Electrolyte System for Small RNA Isolationand Concentration Using ITP in Microchannels Loaded with Pluronic F-127

Illustrated below is ITP system chemistry for small RNA extraction usingITP in microchannels loaded with Pluronic F-127, which is summarized inTable 1.

TABLE 1 System parameters of the leading and trailing electrolytesMobility Conductivity Ionic strength pH (m²V⁻¹s⁻¹) (S m⁻¹) (M) LE^(a)4.0 −68 × 10⁻⁹ 0.86 1.0 × 10⁻¹ TE^(b) 4.0  −4 × 10⁻⁹ 0.04 7.1 × 10⁻³^(a)140 mM 6-aminocaproic acid and 100 mM HCl ^(b)10 mM 6-aminocaproicacid and 50 mM caproic acid

The selection of the electrolyte system is crucial since this allows usto selectively extract and isolate 5 small RNAs from a large variety ofother biomolecules present in the cell lysate, including but not limitedto pre-miRNAs, mRNAs, rRNAs, DNAs, and proteins. The mobility of smallRNAs in our sieving matrix decreases with increasing number ofnucleotides. We have searched for and found an effective mobility forour TE which is between the mobility of small RNAs and pre-miRNAs in thesieving matrix, hence separating these species. Again, Table 1 shows thesystem parameters of the LE and TE; these were determined fromsimulations using Peakmaster 5.2 for the buffer calculations (includingionic strength dependence).40 The current density was typically˜2.45×104 A m-2, and this gave sufficient buffering capacity. Theminimal ionic strength was ˜8 mM during a 5 min ITP experiment and thereservoir size was 5 μL. Therefore, we estimate a change of pH of lessthan ˜0.2 using the Henderson-Hasselbach equation (L. J. Henderson, Am.J. Physiol., 1908, 21, 173-179).

Example 8 Separation of DNA Ladders Using Transient ITP and SievingAgent

To demonstrate the efficacy of 30% Pluronic F-127, we analyzed theseparation of nucleic acids using tITP. FIG. 8A shows the separation ofa 10-100 bp DNA ladder (with 10 by increments). 8 of the 10 fragmentswere detected without optimization of tITP conditions. According to thesupplier, the 10 bp band appears slightly less intense than the otherbands (hence the high mobility peak 1 in FIG. 8A). Further, we separateda 50-800 bp DNA ladder (with 50 bp increments, and a vector fragment>2kbp) as demonstrated in FIG. 8B, and here resolved 15 out of 17fragments. The 350 bp band is designed by the supplier to be two- tothree-times brighter than neighboring bands (hence we identify theintermediate-mobility peak 7 in FIG. 8B). Peak 15 is likely thelow-mobility 2652 bp DNA fragment. Although the DNA ladders shown inFIG. 2 are not perfectly resolved, these experiments confirm that thesieving matrix imparts a significant dependence of electrophoreticmobility on nucleotide length. Below, we demonstrate how we leveragethis dependence in selective isolations of small RNAs. In summary, FIG.8 shows the separation of DNA ladders in 30% Pluronic F-127 using tITP.(a) 8 out of 10 fragments were resolved from a 10-100 bp DNA ladder. Theinset shows the magnification of peak 3 and 4, measured furtherdownstream. (b) For the 50-800 bp DNA ladder 15 out of 17 fragments weredetected. The LE was 140 mM 6-aminocaproic acid with 100 mM HCl, and theTE was 10 mM Bis-Tris and 50 mM HEPES. The dye was 1×SYBR Green I, andthe electric field strength for separation was 1200 V cm⁻¹. The images(from the suppliers) in (a) and (b) show the separation of the DNAladders in 4% and 2% agarose gels, respectively.

Example 9 ITP-based Extraction and Separation of 22 nt from 66 nt Oligoswith Sieving Agent

FIG. 9 is a line graph that shows ITP-based extraction and separation of22 nt from 66 nt oligos in 30% Pluronic F-127. Experimental details areas described in Example 6. The measured signal is dependent only on the22 nt oligo concentration and independent of the 66 nt oligos. Thefluorescence image shows 100 nM 22 nt oligos which are highly focused byITP in the microchannel. The traces are shifted in time by ˜2.5 s (majorpeaks otherwise approximately line up) and fluorescence intensity tofacilitate their comparison. Here, we separated 22 nt from 66 nt oligos,which consisted of only A, C, and G bases. These species have physicalproperties very similar to small RNAs and pre-miRNAs, respectively. Only22 nt oligos are focused by ITP as shown by our spiking procedure andcontrol experiments. In all of the experiments shown, the mobility ofthe TE is higher than our estimates of mobility for oligos of equal orlonger length than 66 nt. The top trace shows the baseline control casewhere no oligos are present. Adding 100 nM of 66 nt oligos results inonly a small peak (near 145 s), which we attribute to shorter-oligoimpurities from the 66 nt oligo sample (the supplier reports that thesample contains 7% shorter fragments remaining after their purificationof the nominal 66 nt oligo sample from polyacrylamide gelelectrophoresis). Next, the sample with 10 nM 22 nt oligos and 100 nM 66nt oligos shows a dramatically increased peak height near 142 s. Spikingthe 22 nt oligo sample (100 nM 22 nt oligos and 100 nM 66 nt oligos)clearly results in the increase of the peak (now at 138 s) which wetherefore identify as 22 nt oligos. This spiking show that thefluorescence intensity of the peak identified as 22 nt oligos isproportional only to the starting concentration of that species, whichis further confirmed in the bottom trace which is spiked with 100 nM 22nt oligos only.

Example 10 Extraction and Separation of 22 nt Oligos from a 0.1-2 kb RNALadder

This example demonstrates the separation of 22 nt oligos from a 0.1-2 kbRNA ladder. Data (not shown) showed separations similar to those inExample 6. The fluorescence intensity is proportional to the startingconcentration of 22 nt oligos, which focus in the ITP zone between theLE and TE. Note for example the third trace from the top which containedboth 22 nt oligos and 0.1-2 kb RNA ladder. Spiking 22 nt oligos atconcentrations of 10, 100 and then 1000 nM progressively increases theassociated peak intensity (and integrated area) as expected. Weattribute the slight peak near 142 s in the top trace to impurities fromthe RNA ladder.

Example 11 Extraction, Isolation and Preconcentration of Small RNAs from293A Cell Lysate

The present ITP-based technique can extract, separate and preconcentratesmall RNAs from pre-miRNAs and other biomolecules in lysate of 293Acells. FIG. 10 is again a line graph that summarizes the results of thecontrol, extraction, and spiking experiments. For experimental details,see Example 7. We have shifted the traces in time (here by 5 s) forclarity. The experiments shown in FIG. 10 used 100×SYBR Green II, exceptfor the second trace (from the top) which used 100×SYBR Green I. In thesecond and third electropherogram, we show that small RNAs from thelysate of 293A cells (at a concentration of 180 cells μL-1) weredetected with SYBR Green I and II, respectively. SYBR Green I (designedto bind to dsDNA) intercalates into RNA at a lower quantum yield thanSYBR Green II.28. Consistently, the RNA peak in the third trace has a15% higher peak height. Spiking the cell lysate with 100 nM 66 nt oligos(fourth trace) results in the same peak shape and intensity for thepresumed short (near 22 nt) RNA peak, as expected. Perhaps the strongestevidence is offered by the fifth trace which shows an 11.7 fold signalincrease when we spike the lysate with (non-native) 100 nM 22 nt oligos.Clearly, the peak height of the isolated small RNA peak has mobilitybelow 66 nt and nearer to that of 22 nt RNAs. Although not shown here,we have also extracted, isolated and quantitated small RNAs from 10 μgml⁻¹ total RNA samples (processed from embryonic kidney cells (293)),and measured the associated peak heights at ˜255 a.u. (data not shown).We have also performed a series of additional calibration experimentswith individual 15 oligos and RNA ladders from which we conclude thatour ITP extraction process has a cut off near 50 nt as the maximum RNAnucleotide length.

One may approximately quantify the amount of small RNAs in our 293A cellline. The bottommost trace of FIG. 10 was spiked with a known amount of100 nM 22 nt oligos, and this peak is 11.7 higher intensity than theunspiked sample peak of the third trace, as per the expected, peak modeITP physics. We can therefore approximate the concentration of smallRNAs in the original 25 sample to be 8.6 nM. Considering theconcentration of 180 cells μL⁻¹, we obtain 2.9×10⁷ small RNAs per cell.Earlier studies have suggested that the total number of piRNAs be on theorder of a million fold, and miRNAs are expressed at high levels up toseveral ten thousands of copies per cell. The total number of types ofsmall RNAs per cell is not known, but we can deduce it by taking theassumption that small RNAs are present at 6×10⁴ copies per cell. Thisresults in an estimate of roughly 500 different types of small RNAs per293A cell, out of which 122 are known today.

Example 12 Alternative ITP-based Extraction of DNA and/or RNA from CellLysate

22-base oligonucleotides were focused with 280 mM Tris-180 mM NicotinicAcid (pH 8) as the LE and 17 mM Tris-10 mM 3.5 Dihydroxybenzoic Acid (pH8) as the TE. TE mM TRIS-10 mM 3.5 DHB were added at the west well (aslaid out in FIG. 7). RNA was added to the West well. The LE was 280 mMTris-180 mM nicotinic acid, The LE and TE are designed to focus onlyshort (approximately <200 base) RNA and no other species. Thetheoretical effective mobility values of the LE and TE are 35×10-9 and33×10-9 m2 V-1 s-1, respectively. This demonstration experiment wasperformed by lysing Pichia pastoris yeast cells and spiking the lysatewith fluorescently labeled 22-base oligonucleotides. We spiked thelysate which had a starting concentration of 60 cells/μL (and a 20 μLsample volume, or approx. 1200 cells) with oligonucleotides, known tohave similar physical properties as short RNA. The LE is 280 mM Tris-180mM Nicotinic Acid (pH 8) and the TE is 17 mM Tris-10 mM 3.5Dihydroxybenzoic Acid (pH 8). In both LE and TE, 0.25% poly(vinyl-pyrrolidone) is present to dynamically suppress EOF but not actas a sieving matrix.

Example 13 Alternative An-chip Separation of DNA from a Mixture of 5Proteins (Using Protein Denaturation)

This experiment shows successful DNA separation from standard proteins,which are major components of genetic samples and major contaminants insample preparation. In this case, the sample was comprised of 5 proteins(Glucose oxidase, Trypsin inhibitor, Myoglobin, Trypsinogen andCytochrome C) labeled with Alexa Fluor 488 (Invitrogen), and 1.5 kbp DNAthat was amplified by polymerase chain reaction from Escherichia colistrain K12. 3 mM salicylic acid and 3 mM valeric acid were added to thesample as spacers in order to separate DNA, proteins, andfree/dissociated fluorophore from proteins as demonstrated. DNA wasintercalated with SYBR Green I (Invitrogen) for fluorescence detection.Proteins in the sample were denatured with sodium dodecyl sulfate and aheat treatment of 95° C. during 10 minutes in order to achieve uniformmobility of all proteins. 100 mM Tris (tris(hydroxymethyl) aminomethane)-50 mM HCl, and 25 mM Tris-192 mM glycine were selected as LEand TE, respectively. The sample was injected between the LE and TE upto a length of 3 mm into the microchannel (70 μm wide and 10 μm deep),and then the electric field of 200 V/cm was applied. The fluorescencewas measured at different times, and DNA after a time showed a peakbetween the proteins (closer to the TE) and the free fluorophore (closerto the LE.

Other protein denaturation agents besides SDS may be used, such as heat,or pH or salts such as urea or guanidine salts or reducing agents whichbreak disulfide bonds. The peptide backbone is left intact.

Example 14 MicroRNA Extraction Using Suppression of Electroosmotic Flow

MicroRNAs are small RNA molecules (˜18 to 24 nucleotides) that canregulate the expression of genes by binding to messenger RNA. Thesemolecules can be specifically focused by ITP in which the LE and TE havea small mobility difference, and focusing inequalities are met. Using280 mM Tris-180 mM nicotinic acid at pH 8 as LE, and 17 mM Tris-10 mM3.5-dihydroxybenzoic acid at pH 8 as TE, their co-ion mobilities arewithin a few percent of each other. To suppress electro-osmotic flow,0.25% poly(vinylpyrrolodine) was added to the LE and TE. Oligos with ˜18to 24 nucleotides have similar physical properties as microRNAs and areinvestigated here. FIG. 11A shows 10 nM 22-base random sequence (A, C,G) oligonucleotides (labeled with Alexa Fluor 488) focused between theLE and TE at an electric field of 190 V/cm. The same molecules at aconcentration of 10 nM were spiked in yeast cell lysate (pichiapastoris) of 60 cells per μl, and extracted using ITP as demonstrated inFIG. 11B. The microchannel inside the glass chip had a width of 90 μmand a depth of 20 μm. For reproducibility, the images were reversed.

Example 15 CO₂ Aided Extraction of 25 bp Ladder from GFP and CAN

FIG. 12 shows a set of isotachopherograms of atmospheric-C02-aidedsimultaneous preconcentration and separation of 25 bp DNA ladder fromgreen fluorescent protein (GFP) and allophycocyanin (ACN) in a singleinterface ITP experiment with 50 mM Tris HCl as the LE and 100 mM Trisglycine as the TE. The electropherograms are obtained 4 s apart and theseparated analyte peaks remain focused and dispersion-free as theymigrate downstream.

Example 16 Introduction of Counterions in a Multiple Well System

FIG. 13 shows a dual-counterion ITP system where both LE wells containthe same leading ion, but different counterions. The applied voltages V₁and V₂ control the ratios of the two counterions in the separationchannel. More wells allow additional degrees of freedom.

For example, the two LE wells shown in the figure control the pH of theTE. Alternatively, the two wells can be configured to control the pH ofthe LE. A third LE well allows control of both simultaneously.Additional wells allow control of, for example, the concentration of asieving matrix or complex-forming counterion. FIG. 13 illustrateschannel bifurcation in which analytes can be distributed to variouschannels. It can be envisioned that a number of bifurcations orjunctions can be designed to distribute different fractions intodifferent channels, using the basic design of FIG. 13. For example, thetarget nucleic acid can be separated from protein, which goes into onechannel and small molecules, which go in to another channel.

CONCLUSION

The above specific description is meant to exemplify and illustrate theinvention and should not be seen as limiting the scope of the invention,which is defined by the literal and equivalent scope of the appendedclaims. Any patents or publications mentioned in this specification areintended to convey details of methods and materials useful in carryingout certain aspects of the invention which may not be explicitly set outbut which would be understood by workers in the field. Such patents orpublications are hereby incorporated by reference to the same extent asif each was specifically and individually incorporated by reference andcontained herein, as needed for the purpose of describing and enablingthe method or material referred to.

What is claimed is:
 1. An isotachophoretic method for extracting atarget nucleic acid from a sample containing a mixture ofmacromolecules, comprising the steps of: (a) treating a sample thatcontains at least one of cells or a target nucleic acid bound to proteinwith at least one of (i) a cell lysis agent, if the target nucleic acidis contained in cells; or (ii), if the target nucleic acid is bound toprotein, with a release agent to release the target nucleic acid fromthe protein, said treating the sample resulting in obtaining a treatedsample containing the target nucleic acid and the mixture ofmacromolecules; (b) causing the treated sample obtained from step (a) tobe present with the target nucleic acid and the mixture ofmacromolecules in a sample well connected to a liquid channel; (c)contacting the treated sample containing the target nucleic acid and themixture of macromolecules with a trailing electrolyte (“TE”) havingmobility greater than said macromolecules that are not target nucleicacid and a mobility less than said target nucleic acid; (d) moving thetreated sample with TE from step (c) to the liquid channel, said liquidchannel containing a leading electrolyte (“LE”) that has a mobilitygreater than said target nucleic acid, wherein said LE and TE containelectrolytes in free solution, and are at pH between about 4 and 10; and(e) applying a voltage along the liquid channel containing said treatedsample, LE, and TE to cause extraction of said target nucleic acid fromsaid macromolecules, in an isotachophoresis interface between LE and TE.2. The method of claim 1 wherein said sample well and liquid channel arecomprised in a microfluidic device.
 3. The method of claim 2 furthercomprising the step of quantifying concentrated target nucleic acid inthe microfluidic device.
 4. The method of claim 1 further comprising thestep of adding a polymer sieving agent in the liquid channel prior toapplying said voltage, to change mobility of one or both of targetnucleic acids and molecules not to be isolated.
 5. The method of claim 4where the sieving agent is selected from the group consisting of blockcopolymer, linear polymers, and cross-linked polymers.
 6. The method ofclaim 1 further comprising the step of adding an agent to the LE priorto applying said voltage for suppressing electroosmotic flow to theliquid channel.
 7. The method of claim 6 where the agent for suppressingelectroosmotic flow is selected from the group consisting ofpolylactams, substituted polyacrylamide derivatives, water solublemethylhydroxyethyl derivatives of cellulose, polyvinylalcohol, andpolyethyleneglycols.
 8. The method of claim 7 where the polylactam ispolyvinylpyrrolidone.
 9. The method of claim 1 where the liquid channeldivides at channel bifurcations to distribute various contents of asample mixture to various channels.
 10. The method of claim 1 where thecell lysis agent is a red blood cell lysis reagent.
 11. The method ofclaim 1 where the release agent is a proteinase.
 12. The method of claim11 where the proteinase is proteinase K.
 13. The method of claim 1wherein said contacting the treated sample from step (b) with a trailingelectrolyte (“TE”) comprises mixing the sample with the TE.
 14. Themethod of claim 1 further comprising the step of removing concentratedtarget nucleic acid from the isotachophoresis interface between LE andTE.
 15. The method of claim 14 wherein said target nucleic acid genomicDNA.
 16. An isotachophoretic method for concentrating small RNA from asample containing a mixture of macromolecules and longer RNA,comprising: (a) obtaining a sample, and if the sample contains cells,treating the sample with a cell lysis agent, to obtain a samplecontaining the macromolecules, small RNA and longer RNA; (b) causing thesample from step (a) to be present with macromolecules, small RNA andlonger RNA in a sample well connected to a liquid channel; (c)contacting the sample of step (b) containing the macromolecules, smallRNA and longer RNA with a trailing electrolyte (“TE”) having mobilityless than said small RNA; (d) moving the sample to a liquid channel witha leading electrolyte (“LE”) that has a mobility greater than said smallRNA, wherein said LE and TE contain electrolytes in free solution, andare at a pH which causes effective mobility of proteins to be differentfrom that of the small RNA; (e) adding a sieving agent to the LE; and(f) applying a voltage along the liquid channel containing said sample,LE and TE to cause extraction of the small RNA from the mixture ofmacromolecules in an isotachophoresis interface between LE and TE. 17.The method of claim 16 wherein step (a) further comprises the step oftreating the sample with an RNAse inhibitor.
 18. The method of claim 16wherein the sieving agent is a polymer.
 19. The method of claim 18wherein the polymer is block copolymer of ethylene oxide and propyleneoxide.
 20. The method of claim 16 wherein step (a) further comprises thestep of treating the sample with one or more of DNAse and proteinase.21. The method of claim 20 wherein the proteinase is proteinase K. 22.The method of claim 16 further comprising the step of adding a proteindenaturing agent to the sample before being injected in the liquidchannel.
 23. The method of claim 16 wherein said contacting of thesample from the sample well with a trailing electrolyte (“TE”) comprisesmixing the sample with the TE.
 24. The method of claim 16 wherein saidLE and TE are at a pH which causes effective mobility of proteins to belower than that of the small RNA.